The availability of quality, affordable sequencing offered by next generation sequencing has revolutionised our understanding of the diversity and importance of the human microbiome to health, and particularly to cardio- metabolic status. There are relatively few studies of the microbiome in Africa, and even fewer examining association with cardio- metabolic health. Many African countries going through transition of lifestyle (food, physical activity), now find cardio-metabolic disorders (obesity, type-2 diabetes) a serious health challenge, and it is important to understand how the microbiome is associated with these disorders. The relationship between health, human genetics, environment and the microbiome is complex. Dugas et al. (2016) have recently called for the ?development of large, globally-distributed, human microbiota studies? to understand the role of the microbiome in epidemiological transition. The AWI-Gen consortium is well placed to explore the role of the microbiome in epidemiological transition. We have six sites in four African countries: Nanoro in Burkina Faso, Navrongo in Ghana, Nairobi in Kenya, and Agincourt, Dikgale and Soweto in South Africa. These sites have different physical environments with significant genetic diversity and are at very different places in epidemiological transition. Soweto in Johannesburg, South Africa reflects a society, which has been urbanized for several generations and in which people have a westernised lifestyle. There are high rates of obesity and cardio-metabolic disorders. The Nanoro and Navrongo sites in Burkina Faso and Ghana are rural communities, where epidemiological transition has just started and people live traditional lifestyles. Levels of obesity are low ? indeed many participants in our studies have very low BMI. Further strengths of AWI-Gen as a lens to explore the relationship between microbiome status and health status are: all participants in phase 1 of AWI-Gen will be genotyped on a high quality chip array; they were finely phenotyped in phase 1 of AWI-Gen and will again be phenotyped in phase 2; and we have good human and equipment infrastructure for the study. This project will explore four key questions: ? What is the diversity of the microbiome in African populations at different stages of epidemiological transition? We will be able to explore this question by examining differences at different sites. ? What is the relationship between microbiome status and cardio-metabolic health? Our studies in phase 1 and 2 of AWI-Gen will collect detailed information about cardio-metabolic health and lifestyle of our participants, and we will be able to find associations with microbiome status. ? How does the microbiome change over time and particularly for women going through hormonal transition? A component of the study will compare pre- and post-menopausal women. The major contribution of AWI-Gen will be to collect base-line data that can be used for long term longitudinal studies. We will also be powered to compare women in different age groups. ? What is the relationship between host genome and microbiome? Recent work has shown that the host genetics can shape the microbiome. As all our participants will be genotyped we can explore this question. Due to sample size limitations, rather than doing a genome-wide study we will focus on replication and genes of specific interest. A further important objective of the study is to lay the basis for future longitudinal studies of how the microbiome changes at both the individual level and the community level. These are important questions that can be explored by us, and others, past the AWI-Gen phase 2 and help sustain future research. There are two important restrictions on our sampling, compromises to ensure sufficient power to answer our hypotheses. (1) Although we will recruit both men and women, we focus on women. Project 3 specifically looks at the effect of hormonal transition and we want to be powered to explore the effect of the microbiome. Also, at several sites women appear to be much more at risk of having high BMI. (2) Infectious diseases such as HIV and malaria are endemic at some sites and can have a profound effect on the microbiome (and perhaps vice- versa). However, in order to reduce variability we will treat these as covariates to be controlled, rather than specifically examining these in hypotheses. The strength of our proposal is that we will be in a good position to apply to other funders for additional funding for nested projects to explore this and other questions within the time-frame of AWI-Gen Phase 2 and beyond.